Many problems in analytical chemistry, and particularly in analytical chemistry for biomedical applications, involve the determination of only a few individual substances (analytes) in very complex samples, such as biological fluids or tissue homogenates. These samples typically contain thousands of individual compounds that are of no interest to the research problem at hand. In addition to the complexity of the samples, the mount of sample volume is often rather limited, particularly in experiments involving laboratory animals. As such, it is often necessary to measure amounts of individual analyte compounds in the picomole range and below. To detect the existence of these compounds of interest, and quantify their presence, an extremely sensitive and selective analytical approach is required.
Recent practice has been to combine existing technologies to achieve the desired end. For example, some analyte samples of interest are particularly susceptible to detection by gas chromatography, mass spectrometry, or some combination thereof. However, many compounds are not readily detectible by either gas chromatography or mass spectrometry. For example, many nonvolatile and thermally labile metabolites of biomedical interest are not directly suitable for gas chromatography/mass spectrometry based analysis. In recent years liquid chromatography/mass spectrometry has overcome several of these problems. However, this technique remains extremely costly and is not suitable for many purposes.
To overcome these shortcomings, researchers often turn to various electrochemical detection systems, and more particularly, to the coupling of liquid chromatography, with electrochemistry. Another technique of interest is to combine in vivo membrane sampling with liquid chromatography/electrochemistry to monitor compounds continuously extracted from living organisms such as laboratory animals.
Combining a liquid chromatography separation system with an electrochemistry detection system to create a combined liquid chromatography/electrochemistry (LCEC) detection system is well known. The basic outline of a conventional LCEC analyzer is shown schematically in FIG. 1.
An LCEC analyzer 10 includes a pump 12 for pumping a mobile phase (such as that contained in flask 14) through a tube 16. Preferably, the pump 12 used for pumping the mobile phase is a constant flow, reciprocating dual piston pump. The pump should provide a flow that is as smooth and pulseless as possible, thereby minimizing baseline noise. An injection valve 20 is provided for injecting the sample into the mobile phase that is flowing through the tube 16. The sample/mobile phase mixture then enters the upstream end 26 of a liquid chromatography column 24, passes through the column 24, and emerges at the downstream end 30 of the column 24. In order to maintain temperature control, the chromatography column 24 is mounted to a heater block 25.
The material that enters the upstream end 26 of the column 24 is very different than that which emerges from the downstream end 30. At the upstream end 26, the sample comprises a sample mixture, wherein all of the various analytes are mixed within the sample. As the sample flows down the column 24, the various analytes contained within the sample migrate at various rates, so that at the downstream end 30 of the column 24, the constituent analytes emerge in generally discrete, separated analyte bands.
Following the separation of components, the eluent (containing both mobile phase and analytes) is conducted through an analyte transfer tube portion 32 to an electrochemical detector cell 34. The electrochemical detector cell 34 typically includes a working electrode, an auxiliary electrode and a reference electrode. A potential is supplied at the working electrode interface by an amperometric controller 36 that also measures the resulting electrolysis current across the sample within the electrochemical cell 34. A recorder 38 is provided for recording the output of the controller 36. Typically, this output consists of a chart graph of electrical current as a function of time. The heated metal block 25 helps to ensure uniformity, and reproduceability of results by controlling the temperature of the mobile phase and the sample flowing through the column 24 and the detector cell 34.
Electrochemical detection is based on a controlled potential and a measured electrolysis current. A predetermined potential difference (usually between (+1.4) and (-1.5) volts, and dependent upon the redox behavior of the analyte to be detected) is applied between the reference and the working electrodes. The applied potential serves as the driving force for the electrochemical reaction that occurs in the detector cell, at the working electrode surface. As the potential of the working electrode relative to the reference electrode becomes more positive, the surface (of the working electrode) becomes a better oxidizing agent (electron sink). Conversely, the more negative the applied potential, the better the working electrode acts as a reducing agent (electron source).
As an oxidizable solute (e.g. norepinephrine) passes over the surface of the working electrode, those molecules immediately adjacent to the electrode surface will be oxidized in a heterogeneous transfer of electrons. The current that results from this exchange of electrons with the surface is monitored as a function of time. Since the rate of material conversion by the electrochemical reaction is proportional to the instantaneous concentration, the current will be directly related to the amount of analyte eluted as a function of time. If the chromatographic condition (mobile phase, flow rate, temperature, etc.) are carefully controlled, amperometric detection can be quite precise, permitting the user to obtain quantitative data at the picomole level (total injected amount) for many compounds.
A wide variety of working electrodes can be used in the electrochemical detector. The electrode used should be physically and chemically inert to the mobile phase at the chosen applied potential. Four electrode surfaces that have been found to be useful are glassy carbon, carbon composites, platinum and mercury. Glassy carbon is versatile because it has excellent resistance to nearly any solvent used in liquid chromatography, and may be used over a wide potential range. Carbon composites have been used as a reliable surface for many years for the determination of catecholamines and related substances. Platinum is especially useful for the determination of hydrogen peroxide. Mercury provides an extended negative range of potential, but has very limited applications when using positive potential.
An early example of an LCEC cell was proposed by Fleet and Little. See, B. Fleet and C. J. Little, J. Chromatography 12 (1974) at pp. 747-752. In the Fleet and Little cell, eluent is directed as a "jet" onto a single working electrode. The Fleet and Little cell is an example of a "wall jet" type cell wherein a probe-like jet having a relatively smaller diameter is positioned to direct eluent onto a relatively larger diameter working electrode. To achieve wall jet characteristics, there must be sufficient volume to permit the eluent that is directed onto the electrode to flow radially and axially away from the point of impact on the electrode.
An example of another known prior art electrochemical cell is shown in Shoup, Bruntlett, Jacobs, and Kissinger, "LCEC: A Powerful Tool For Biomedical Problem Solving", American Laboratory, October, 1981. This article discloses a thin-layer amperometric transducer for use in liquid chromatography/electrochemistry applications. The device shown in the Shoup article uses a thin layer "cross-flow" pattern, wherein the sample enters the detector cell at one side of a working electrode, flows across the working electrode to the other side of the working electrode, and then flows out through an outlet robe. Once the material flows out the outlet tube, it passes over a reference electrode, and then is lead to a collection tube. Electrochemical detector cells of the type described above have been manufactured and marketed since 1974 by Bioanalytical Systems, Inc. of West Lafayette, Ind., the assignee of the instant invention.
An improved, later generation thin-layer electrochemical detector for use in LCEC applications is shown in Kissinger, "Biomedical Applications of Liquid Chromatography-Electrochemistry", Journal of Chromatography, Number 488 (1989) at pages 31-52. The device discussed in this article was invented by Ronald Shoup, a co-worker of the applicants in 1985, and is manufactured by Bioanalytical Systems, Inc. of West Lafayette, Ind., the assignee of the present invention. The Shoup device includes an auxiliary electrode block, and a working electrode block. Between the two blocks is a gasket which, when assembled includes a "cross-flow" path defined between the surfaces of the auxiliary and working electrode blocks, and the interior "cut out" portion of the gasket. Sample exiting a liquid chromatography column flows through an analyte transfer tube and around a mobile phase preheater for maintaining a desired temperature. The sample then passes through an inlet in the auxiliary electrode onto a working surface of a working electrode block. The analyte then flows linearly across the working electrode to the opposite side of the working surface of the working electrode block, and then out the outlet of the block. One of the very useful features of the Shoup device is that it includes a clamp system to facilitate replacement of the electrodes in the device.
A wide variety of working electrode configurations can be used with both flow cells described above, including a single working electrode, a dual parallel electrode, and a dual series electrode. Electrochemical detector cells of the type described above are suitable for a wide range of applications, have proven to be great commercial successes and are widely accepted by the scientific community.
However, room for improvement still exists. This need for improvement is driven largely by the desire to perform analytical operations on smaller and smaller sample volumes. Originally, the electrochemical detector cells described above were used with chromatography columns that were between about 3 and 10 millimeters in diameter, and through which liquid flowed at rates from about 1 to 5 milliliters per minute. Today, liquid chromatography has evolved in which "microbore" chromatography columns are used having an internal diameter of 1 millimeter or less. Some chromatography columns even have internal diameters of several tens of micrometers. Examples of such microbore chromatography columns are the SEPSTIK series of chromatography columns manufactured by Bioanalytical Systems, Inc.
The use of such small volumes of sample material is not always one of choice. In biomedical applications, compelling advantages exist for using much smaller volumes of material. For example, certain biological systems may only be able to yield very small quantities of fluid. Other situations exist wherein it is desirable for the organism to only withdraw such small volumes of fluid.
Because of the small size of the columns, the flow rate through these microbore chromatography columns is dramatically reduced, as compared to the flow rates used for traditional liquid chromatography columns. For example, many of these microbore chromatography columns have internal diameters of about 1 millimeter and typically utilize flow rates therethrough on the order of between about 50 and 200 microliters per minute of material.
As a result of these small flow rates, the volume in which the sample analytes are contained as they elute from the liquid chromatography column is much smaller. Because of this, conventional electrochemical detector devices must be improved to accommodate these smaller volumes without distorting the concentration profiles for the substances eluting from the column. It has been found by the applicants that the "cross-flow" designs used in the two detector cells discussed above can be improved upon for detecting analytes at these small volumes and flow rates.
It is therefore one object of the present invention to provide an electrochemical detector cell that has utility when used to detect the presence and quantity of analytes in very small sample volumes. It is also an object of the present invention that such detection be done in a reliable and repeatable manner by the detector cells.
A related problem experienced with some, prior art detectors is the amount of "dead volume" in the analyte transfer tube portion of the LCEC detector between the downstream end 30 of the liquid chromatography column 24, and the working electrode of the detector cell 34. It is preferable to minimize dead volume space. In this dead volume space, the sharp bands of analyte that emerge from the downstream end 30 of the liquid chromatography column 24 have a tendency to spread out, and become less defined and concentrated as the material moves through the dead volume. The amount of definition loss for the separated bands that occurs is related to the amount of dead volume. Although this distortion of the bands is not critical when using relatively larger volumes of fluid, it becomes much more critical when the volume that contains the analytes and the flow rate are smaller.
It is therefore also one object of the present invention to provide an electrochemical detector cell that minimizes dead volume between a liquid chromatography column and the working electrode.
Another way in which known electrochemical detector cells can be improved is to make the cells compatible with arrangements wherein multiple detector cells are used. Often a sample of interest will contain a variety of analytes whose presence and quantity are desired to be determined. In many situations, not all of the analytes of interest can be determined using only one electrode, or using only one detector cell. For example, some analytes must be detected using an oxidation reaction, whereas others must be detected using a reduction reaction. In such cases, a single electrode, or a single detector may not be capable of detecting both the oxidation-requiring analyte, and the reduction-requiring analyte. To do so requires either a series of electrodes, or a series of detector cells.
One difficulty with some known prior detectors is that they generally were not well suited to being used in series with other detectors. Many were not suitable because they contained a large volume area into which the analyte flowed. For example, wall jet type electrodes usually required a relatively large volume area immediately downstream in the flow path from the working electrode. Others used a liquid junction reference electrode that required a large volume area.
When an analyte flows into such a large volume, the analyte material tends to mix with other analytes in the sample in the large volume, thus disbursing the nice, relatively "tight" band of analyte that emerged from the liquid chromatography column.
It is therefore also an object of the present invention to create an electrochemical detector cell that helps to better preserve band integrity of an analyte during the passage of a band of analyte through the detector cell, and thereby improve the ability of the detector to be used in an arrangement that includes a series of detector cells.
In addition to its use with liquid chromatography columns, the electrochemical detector cell the present invention also has utility in connection with microdialysis and ultrafiltration in "in-vivo" sampling systems. Microdialysis sampling systems typically involve the use of a probe that can be placed into a living organism, such as a brain, blood vessel, duct or other tissue. The microdialysis probe typically includes a tube having a selectively permeable membrane, through which the body fluid of interest can pass. A perfusion fluid is flowed into the probe past the dialysis membrane to help pick up molecules diffusing through the wall of the selectively permeable membrane. The constant flow of the perfusion fluid through the probe creates a concentration gradient across the dialysis membrane. Chemicals in the extracellular fluid of the surrounding tissue will diffuse across this membrane under the influence of this gradient. Chemicals can also flow from the perfusion fluid into-the tissue according to the same principle.
A technique related to microdialysis is ultrafiltration. Ultrafiltration is generally similar to microdialysis. However, instead of the use of a concentration gradient, fluid is extracted from the surrounding tissue by the application of a vacuum to the dialysis membrane.
Both microdialysis and ultrafiltration probes can be placed into living tissue to study the metabolism of a conscious, moving animal. Determination of the concentration and variety of small molecules in a microdialysis or ultrafiltration sample is then possible, to permit the user to obtain a "real time" analysis of the concentration of analyte within the living organism. For example, a rat can be fed a material, such as the drug acetaminophen. Brain tissue can then be sampled through microdialysis to determine the time required for the acetaminophen to move from the animal's digestive tract to a particular part of the animal's brain. Through studies such as this, the efficacy of a drug can be studied and determined. Once the material is removed from the microdialysis or ultrafiltration probe, it can be transferred to an electrochemical detector cell, wherein the presence and quantity of the analyte of interest can be detected.
One difficulty with detecting such materials is that the volume of sample and analyte withdrawn from a living organism is typically also very small, usually only a few microliters. It is therefore also one object of the present invention to provide an electrochemical detector cell that is well suited for use with the small volumes of analyte available in connection with microdialysis and ultrafiltration techniques.